Our work in this area has three broad areas of focus: (1) Development of rapid nanopore-based strategies to sequence bacterial resistance plasmids with clinical diagnostic applications; (2) Use of these sequencing strategies to characterize genomic mechanisms of antimicrobial resistance in clinical isolates, and their emergence; (3) Application of RNA-seq to study gene expression from resistance plasmids. These areas of focus seek both to evaluate sequencing methods that can be used practically in a clinical microbiology lab and to study the biology of plasmid-based mechanisms underlying antimicrobial resistance. (1) Nanopore sequencing methods provide ultrafast, read-by-read data availability that may be used to support real-time infectious disease diagnostics, including rapid sequencing of bacterial isolates and resistance gene identification in the clinical microbiology lab. Work done during the current fiscal year developed a practical nanopore sequencing workflow that takes as input purified plasmid DNA and can produce full annotations of all plasmid-based resistance genes within a working day, starting with subcultured isolates (manuscript submitted). Current work is in progress to extend these methods to allow sequencing of resistance plasmids from single bacterial colonies from primary cultures. (2) The evolution of pathogenic MDR phenotypes in the natural in vivo context of acute and chronic infection is being studied through genomic analysis of serially-collected human clinical bacterial isolates. Four initial isolate collections have been assembled along with with comprehensive microbiologic characterization, with a focus on evolution of MDR phenotypes in pathogenic non-fermenting gram negative proteobacteria (Pseudomonas, Burkholderia, Achromobacter, and Bordetella). Work done during this fiscal year demonstrated the role that hypermutation may play in facilitating the emergence of resistance to ceftazidime-avibactam in P. aeruginosa (Khil et al, ASM 2017; manuscript in preparation). Work in the next fiscal year will examine pathoadaptation and emergence of resistance phenotypes in Bordetella, Achromobacter and Burkholderia. (3) Global gene expression analysis using NGS technology (RNA-seq) offers a powerful approach to study the large-scale structure of transcription and transcriptional regulation. In these experiments, we are employing strand-specific methods to study gene expression from resistance plasmids from clinical bacterial isolates. Both context-dependence of expression and regulatory antisense RNA is being studied with this technique. In particular, we are investigating the consequences of large scale insertion-sequence mediated rearrangements in plasmids that have occurred in vivo, studied previously with bioinformatics approaches in He et al, 2016. Work done during the past and current fiscal year has produced rich RNA-seq data sets from plasmids from a number of clinical isolates, currently under analysis.